Regulation of hepatic differentiation of human embryonic stem cells by calcium silicate extracts for liver injury repairing

Abstract Directing embryonic stem cell (ESC)-derived hepatocytes is critical in understanding hepatic differentiation and applying cell-based treatment to severe liver diseases. While growth factor-based strategies are widely used, using chemical cues could present an alternative to optimize the strategies for stem cell differentiation. Here, for the first time, an inorganic calcium silicate (CS, CaSiO3)-based approach, together with a modified four-stage differentiating protocol, was proposed to quantify the effects of CS extracts on inducing hepatic differentiation of human ESCs (H9 cells). The roles of CS-activated H9 cells in liver injury repair were tested by cell tracking and immunohistochemical staining. Results indicated that high concentrations of CS extracts initially enhance definitive endodermal (DE) lineage, followed by gradual DE differentiation at low CS concentrations. The order of CS addition is also crucial, since the presence at stemness stage and the absence at DE stage could optimize hepatic differentiation capacity of H9 cells, resulting in optimized cells that differentiate into functional hepatocyte-like cells. The addition of CS extracts at precursor hepatocyte stage enhances their maturity, which favors the turnover of liver injury in CCl4-treated mice. These results provide an insight into applying bioactive inorganic biomaterials to foster hepatic differentiation of human ESCs for cell therapy.